The invention relates generally to the identification of inhibitors of ATP synthase (F1/F0 ATPase), useful as antimicrobial or anti-proliferative agents.
Infectious diseases can be caused by a number of organisms, including bacteria, fungi, protozoans and other parasites, and viruses. Bacteria as a group generally include gram-negative bacteria, gram-positive bacteria, spirochetes, rickettsiae, mycoplasmas, mycobacteria and actinomycetes. Resistance of bacteria and other pathogenic organisms to antimicrobial agents is an increasingly troublesome problem. The accelerating development of antibiotic-resistant bacteria, intensified by the widespread use of antibiotics in farm animals and overprescription of antibiotics by physicians, has been accompanied by declining research into new antibiotics with different modes of action. [Science, 264: 360-374 (1994)].
Antibacterial agents can be broadly classified based on chemical structure and proposed mechanism of action, and major groups include the following: (1) the xcex2-lactams, including the penicillins, cephalosporins and monobactams; (2) the aminoglycosides, e.g., gentamicin, tobramycin, netilmycin, and amikacin; (3) the tetracyclines; (4) the sulfonamides and trimethoprim; (5) the fluoroquinolones, e.g., ciprofloxacin, norfloxacin, and ofloxacin; (6) vancomycin; (7) the macrolides, which include for example, erythromycin, azithromycin, and clarithromycin; and (8) other antibiotics, e.g., the polymyxins, chloramphenicol and the lincosamides.
Antibiotics accomplish their anti-bacterial effect through several mechanisms of action which can be generally grouped as follows: (1) agents acting on the bacterial cell wall such as bacitracin, the cephalosporins, cycloserine, fosfomycin, the penicillins, ristocetin, and vancomycin; (2) agents affecting the cell membrane or exerting a detergent effect, such as colistin, novobiocin and polymyxins; (3) agents affecting cellular mechanisms of replication, information transfer, and protein synthesis by their effects on ribosomes, e.g., the aminoglycosides, the tetracyclines, chloramphenicol, clindamycin, cycloheximide, fucidin, lincomycin, puromycin, rifampicin, other streptomycins, and the macrolide antibiotics such as erythromycin and oleandomycin; (4) agents affecting nucleic acid metabolism, e.g., the fluoroquinolones, actinomycin, ethambutol, 5-fluorocytosine, griseofulvin, rifamycins; and (5) drugs affecting intermediary metabolism, such as the sulfonamides, trimethoprim, and the tuberculostatic agents isoniazid and para-aminosalicylic acid. Some agents may have more than one primary mechanism of action, especially at high concentrations. In addition, secondary changes in the structure or metabolism of the bacterial cell often occur after the primary effect of the antimicrobial drug.
Protozoa account for a major proportion of infectious diseases worldwide, but most protozoan infections occur in developing countries. Treatment of protozoan infections is hampered by a lack of effective chemotherapeutic agents, excessive toxicity of the available agents, and developing resistance to these agents.
Fungi are not only important human and animal pathogens, but they are also among the most common causes of plant disease. Fungal infections (mycoses) are becoming a major concern for a number of reasons, including the limited number of antifungal agents available, the increasing incidence of species resistant to known antifungal agents, and the growing population of immunocompromised patients at risk for opportunistic fungal infections, such as organ transplant patients, cancer patients undergoing chemotherapy, burn patients, AIDS patients, or patients with diabetic ketoacidosis. The incidence of systemic fungal infections increased 600% in teaching hospitals and 220% in non-teaching hospitals during the 1980""s. The most common clinical isolate is Candida albicans (comprising about 19% of all isolates). In one study, nearly 40% of all deaths from hospital-acquired infections were due to fungi [Sternberg, Science, 266:1632-1634 (1994)].
Known antifungal agents include polyene derivatives, such as amphotericin B (including lipid or liposomal formulations thereof) and the structurally related compounds nystatin and pimaricin; flucytosine (5-fluorocytosine); azole derivatives (including ketoconazole, clotrimazole, miconazole, econazole, butoconazole, oxiconazole, sulconazole, tioconazole, terconazole, fluconazole, itraconazole, voriconazole [Pfizer], poscaconazole [SCH56592, Schering-Plough]) and ravuconazole; allylamines-thiocarbamates (including tolnaftate, naftifine and terbinafine); griseofulvin; ciclopirox; haloprogin; echinocandins (including caspofungin [MK-0991, Merck], FK463 [Fujisawa] and VER-002 [Versicor]); nikkomycins; and sordarins. Recently discovered as antifungal agents are a class of products related to bactericidal/permeability-increasing protein (BPI), described in U.S. Pat. Nos. 5,627,153, 5,858,974, 5,652,332, 5,856,438, 5,763,567 and 5,733,872, the disclosures of all of which are incorporated herein by reference.
Bactericidal/permeability-increasing protein (BPI) is a protein isolated from the granules of mammalian polymorphonuclear leukocytes (PMNs or neutrophils), which are blood cells essential in the defense against invading microorganisms. See Elsbach, 1979, J Biol. Chem., 254: 11000; Weiss et al., 1987, Blood 69: 652; Gray et al., 1989, J Biol. Chem. 264: 9505. The amino acid sequence of the entire human BPI protein and the nucleic acid sequence of DNA encoding the protein (SEQ ID NOS: 1 and 2) have been reported in U.S. Pat. No. 5,198,541 and FIG. 1 of Gray et al., J. Biol. Chem., 264:9505 (1989), incorporated herein by reference. Recombinant human BPI holoprotein has also been produced in which valine at position 151 is specified by GTG rather than GTC, residue 185 is glutamic acid (specified by GAG) rather than lysine (specified by AAG) and residue 417 is alanine (specified by GCT) rather than valine (specified by GTT). An N-terminal fragment of human BPI possesses the anti-bacterial efficacy of the naturally-derived 55 kD human BPI holoprotein. (Ooi et al., 1987, J Bio. Chem. 262: 14891-14894). In contrast to the N-terminal portion, the C-terminal region of the isolated human BPI protein displays only slightly detectable anti-bacterial activity against gram-negative organisms and some endotoxin neutralizing activity. (Ooi et al., 1991, J Exp. Med. 174: 649). An N-terminal BPI fragment of approximately 23 kD, referred to as rBPI23, has been produced by recombinant means and also retains anti-bacterial, including anti-endotoxin activity against gram-negative organisms (Gazzano-Santoro et al., 1992, Infect. Immun. 60: 4754-4761). An N-terminal analog designated rBPI21, (also referred to as rBPI(1-193)ala132) has been described in U.S. Pat. No. 5,420,019.
Three separate functional domains within the recombinant 23 kD N-terminal BPI sequence have been discovered (Little et al., 1994, J Biol. Chem. 269: 1865). These functional domains of BPI designate regions of the amino acid sequence of BPI that contributes to the total biological activity of the protein and were essentially defined by the activities of proteolytic cleavage fragments, overlapping 15-mer peptides and other synthetic peptides. Domain I is defined as the amino acid sequence of BPI comprising from about amino acid 17 to about amino acid 45. Initial peptides based on this domain were moderately active in both the inhibition of LPS-induced LAL activity and in heparin binding assays, and did not exhibit significant bactericidal activity. Domain II is defined as the amino acid sequence of BPI comprising from about amino acid 65 to about amino acid 99. Initial peptides based on this domain exhibited high LPS and heparin binding capacity and exhibited significant antibacterial activity. Domain III is defined as the amino acid sequence of BPI comprising from about amino acid 142 to about amino acid 169. Initial peptides based on this domain exhibited high LPS and heparin binding activity and exhibited surprising antimicrobial activity, including antifungal and antibacterial (including, e.g., anti-gram-positive and anti-gram-negative) activity. The biological activities of peptides derived from or based on these functional domains (i.e., functional domain peptides) may include LPS binding, LPS neutralization, heparin binding, heparin neutralization or antimicrobial activity.
Many other utilities of BPI protein products, including rBPI23 and rBPI21, have been described due to the wide variety of biological activities of these products. For example, BPI protein products are bactericidal for gram-negative bacteria, as described in U.S. Pat. Nos. 5,198,541, 5,641,874, 5,948,408, 5,980,897 and 5,523,288. International Publication No. WO 94/20130 proposes methods for treating subjects suffering from an infection (e.g. gastrointestinal) with a species from the gram-negative bacterial genus Helicobacter with BPI protein products. BPI protein products also enhance the effectiveness of antibiotic therapy in gram-negative bacterial infections, as described in U.S. Pat. Nos. 5,948,408, 5,980,897 and 5,523,288 and International Publication Nos. WO 89/01486 (PCT/US99/02700) and WO 95/08344 (PCT/US94/11255). BPI protein products are also bactericidal for gram-positive bacteria and mycoplasma, and enhance the effectiveness of antibiotics in gram-positive bacterial infections, as described in U.S. Pat. Nos. 5,578,572 and 5,783,561 and International Publication No. WO 95/19180 (PCT/US95/00656). BPI protein products exhibit antifungal activity, and enhance the activity of other antifungal agents, as described in U.S. Pat. No. 5,627,153 and International Publication No. WO 95/19179 (PCT/US95/00498), and further as described for BPI-derived peptides in U.S. Pat. No. 5,858,974, which is in turn a continuation-in-part of U.S. application Ser. No. 08/504,841 and corresponding International Publication Nos. WO 96/08509 (PCT/US95/09262) and WO 97/04008 (PCT/US96/03845), as well as in U.S. Pat. Nos. 5,733,872, 5,763,567, 5,652,332, 5,856,438 and corresponding International Publication Nos. WO 94/20532 (PCT/US/94/02465) and WO 95/19372 (PCT/US94/10427). BPI protein products exhibit anti-protozoan activity, as described in U.S. Pat. Nos. 5,646,114 and 6,013,629 and International Publication No. WO 96/01647 (PCT/US95/08624). BPI protein products exhibit anti-chlamydial activity, as described in co-owned U.S. Pat. No. 5,888,973 and WO 98/06415 (PCT/US97/13810). Finally, BPI protein products exhibit anti-mycobacterial activity, as described in co-owned, co-pending U.S. application Ser. No. 08/626,646, which is in turn a continuation of U.S. application Ser. No. 08/285,803, which is in turn a continuation-in-part of U.S. application Ser. No. 08/031,145 and corresponding International Publication No. WO 94/20129 (PCT/US94/02463).
The effects of BPI protein products in humans with endotoxin in circulation, including effects on TNF, IL-6 and endotoxin are described in U.S. Pat. Nos. 5,643,875, 5,753,620 and 5,952,302 and corresponding International Publication No. WO 95/19784 (PCT/US95/01151).
BPI protein products are also useful for treatment of specific disease conditions, such as meningococcemia in humans (as described in U.S. Pat. Nos. 5,888,977 and 5,990,086 and International Publication No. WO97/42966 (PCT/US97/08016), hemorrhage due to trauma in humans, (as described in U.S. Pat. Nos. 5,756,464 and 5,945,399, U.S. application Ser. No. 08/862,785 and corresponding International Publication No. WO 97/44056 (PCT/US97/08941), burn injury (as described in U.S. Pat. No. 5,494,896 and corresponding International Publication No. WO 96/30037 (PCT/US96/02349)) ischemia/reperfusion injury (as described in U.S. Pat. No. 5,578,568), and depressed RES/liver resection (as described in co-owned, co-pending U.S. application Ser. No. 08/582,230 which is in turn a continuation of U.S. application Ser. No. 08/318,357, which is in turn a continuation-in-part of U.S. application Ser. No. 08/132,510, and corresponding International Publication No. WO 95/10297 (PCT/US94/11404).
BPI protein products also neutralize the anticoagulant activity of exogenous heparin, as described in U.S. Pat. No. 5,348,942, neutralize heparin in vitro as described in U.S. Pat. No. 5,854,214, and are useful for treating chronic inflammatory diseases such as rheumatoid and reactive arthritis, for inhibiting endothelial cell proliferation, and for inhibiting angiogenesis and for treating angiogenesis-associated disorders including malignant tumors, ocular retinopathy and endometriosis, as described in U.S. Pat. Nos. 5,639,727, 5,807,818 and 5,837,678 and International Publication No. WO 94/20128 (PCT/US94/02401).
BPI protein products are also useful in antithrombotic methods, as described in U.S. Pat. Nos. 5,741,779 and 5,935,930 and corresponding International Publication No. WO 97/42967 (PCT/US7/08017).
There continues to exist a need for novel antimicrobial agents and anti-proliferative agents and for methods of identifying such novel compounds. Such methods ideally would identify compounds that are unrelated to conventional agents and that target different aspects of cell growth and replication compared to conventional agents.
One aspect of the present invention provides methods for identifying novel compounds that inhibit the function of F1/F0 ATPase (also referred to as ATP synthase or ATP synthetase or F0/F1 ATPase) and that have antimicrobial or anti-proliferative activity, particularly antibacterial activity. Such novel compounds are identified either through screening libraries of existing molecules, such as inorganic or organic compounds (including bacterial, fungal, mammalian, insect or plant products, peptides, peptidomimetics and/or organomimetics) or through rational design of molecules that specifically interfere with the function of the ATP synthase. Novel antimicrobial and/or anti-proliferative compounds identified by such methods are also provided.
It is contemplated that screening methods according to the present invention may involve one or more assays, including an assay for ability of test compounds to inhibit or produce a decrease in the activity of ATP synthase preparations, e.g., as directly measured by ATP hydrolysis or ATP synthesis, or as indirectly measured by oxygen consumption assays, such as changes in the rate of oxygen consumption of mitochondria (e.g., State 3 or State 4) or whole cells, or by assays to detect alterations in electron transport or associated cytochromes, proton gradient, or membrane potential, or by mitochondrial function of whole mitochondria or submitochondrial particles (e.g. by measuring total ATP levels); or an assay for ability of test compounds to interact (including, e.g., ability to bind to or abiltiy to competitively inhibit binding of BPI-derived peptides to) with ATP synthase.
It is further contemplated that screening methods according to the present invention may involve additional screening steps. For example, the screening may include selection of test compounds that have a differential effect on microbial target cells in comparison to other types of cells (e.g., a greater effect on bacterial cells relative to mammalian cells, or a greater effect on bacterial cells relative to fungal cells). Suitable candidate compounds may have a 2-fold or more, 10-fold or more, 50-fold or more, or 100-fold or more separation between target cell activity and other cell toxicity. Final stages of screening for antimicrobial or anti-proliferative agents may include conventional testing for in vitro and/or in vivo activity against a variety of organisms using procedures known in the art.
According to this aspect of the invention, methods are provided for identifying a potential or candidate antimicrobial compound comprising the steps of: (a) selecting a test compound that interacts with or that produces a decrease in the activity of an F1/F0 ATP synthase (e.g., a bacterial cell F1/F0 ATP synthase, or a protozoan/parasite mitochondrial ATP synthase, or fungal or mammalian mitochondrial ATP synthase); and (b) detecting inhibition of growth of microbial target cells in the presence of the selected test compound from step (a). Step (a) can be carried out, for example,. by determining F1/F0 ATP synthase activity in the presence and absence of the test compound followed by selecting test compounds that inhibit or produce a decrease in the activity. Step (b) can be carried out, for example, by detecting growth of microbial target cells in the presence and absence of the test compound, followed by selecting a test compound that inhibits growth.
The F1/F0 ATP synthase need not be, but is preferably from the microbial target cell. Alternatively, a mammalian or other eukaryotic mitochondrial F1/F0 ATP synthase could be used. For example, oxygen consumption of mammalian or other eukaryotic mitochondria can be measured in step (a).
Optionally, another screening step (c) is utilized (instead of, concurrent with, or subsequent to step (b)) that involves detecting a reduction in total ATP levels of the microbial target cell or its mitochondria in the presence of the selected test compound compared to the absence of the test compound. Preferably an additional screening step is utilized that involves detecting growth of a non-target cell (e.g., a mammalian cell) in the presence and absence of the selected test compound.
Another aspect of the invention involves selecting a test compound that produces a decrease in total ATP levels of microbial cells or their mitochondria, relative to ATP levels in the absence of the test compound, followed by an additional screening step such as detecting growth of microbial target cells, and/or detecting growth of non-target cells.
Another aspect of the invention provides methods of identifying antimicrobial compounds by assaying for ability to uncouple electron transport from ATP synthesis, e.g., by measuring oxygen consumption of whole cells, mitochondria or bacterial membrane preparations in the presence and absence of the test compound followed by measuring oxygen consumption after further addition of a known uncoupling agent, preferably at the maximally effective uncoupling concentration of both the test compound and the known uncoupling agent. If the test compound is an uncoupling agent, addition of the known uncoupling agent will produce no significant increase in oxygen consumption. Such assay steps can be combined with other assays disclosed herein and preferably are combined with selection of test compounds that have a differential effect on target cells in comparison to other types of cells.
Specifically provided are the use of such screening methods to identify novel antimicrobial agents that are active against pathogenic organisms that rely on ATP synthase for aerobic metabolism, for example, gram-negative bacteria, gram-positive bacteria, Mycoplasma, Mycobacteria, protozoa, or other prokaryotes. Also specifically provided are the use of such screening methods to identify novel insecticidal agents or novel herbicidal agents that are active against, for example, plant organisms (including weeds or algae).
Another aspect of the invention provides methods for treating infections involving pathogenic organisms that rely on ATP synthase for aerobic metabolism (for example, gram-negative bacteria, gram-positive bacteria, Mycoplasma, protozoa, or other prokaryotes or parasites) by inhibiting the activity of ATP synthase, using compounds other than those presently known in the art, such as BPI protein products or BPI-derived peptides previously known in the art. Concurrent administration of other conventional antimicrobial agents is contemplated.
A further aspect of the invention provides methods for killing or inhibiting growth of insects or plants by inhibiting the activity of their respective ATP synthases. Preferred methods involve administration of BPI protein products, including BPI-derived peptides.
Corresponding screening methods for identifying antifungal agents, as well as novel agents and therapeutic uses made possible by these methods, are addressed in U.S. Provisional Application Ser. No. 60/143,372 filed Jul. 12, 1999 and corresponding co-owned, concurrently filed U.S. application Ser. No. 09/543,802, both of which are incorporated herein by reference. According to yet another aspect of the invention, the discovery that BPI protein products, particularly BPI-derived peptides, also inhibit mammalian mitochondrial ATP synthase activity provides a basis for use of these compounds, alone or in association with appropriate targeting agents, as anti-proliferative or cytotoxic agents that can be used to treat conditions for which an inhibition of cellular proliferation is desirable, including cancer or other neoplastic diseases, autoimmune diseases, etc.
Numerous additional aspects and advantages of the invention will become apparent to those skilled in the art upon consideration of the following detailed description of the invention which describes presently prepared embodiments thereof.